Repeated heat exposure upregulates skeletal muscle aquaporin‐4 expression in male mice

Heat acclimation involves coordinated physiological adaptations, yet the role of skeletal muscle water channels remains insufficiently defined. We investigated how repeated heat exposure affects aquaporin-4 (AQP4) in skeletal muscle using both in vivo and in vitro models. Male C57BL/6NJ mice were exposed to daily passive heating (45°C for 30 min/day, 14 days). Repeated heat exposure reduced body-weight gain and markedly attenuated Hspa1a induction following an acute heat challenge, confirming systemic heat acclimation. Immunofluorescence analysis revealed an approximately 1.3-fold increase in AQP4 protein in fast-twitch muscles without changes in its subcellular localization. In vitro, C2C12 myocytes were subjected to repeated heat bouts (42°C, 3 h/day for 1-5 days). Heat-acclimated cells exhibited blunted Hspa1a induction in response to acute heat stress and progressively increased AQP4 protein levels (approximately 1.6-fold), indicating that heat stress directly promotes AQP4 upregulation in muscle cells. Together, these findings identify AQP4 as a heat-responsive component of skeletal muscle and suggest that AQP4 may contribute to peripheral osmotic regulation during heat acclimation.